Insertable variable fragments of antibodies and modified a1-a2 domains of nkg2d ligands

ABSTRACT

This application relates generally to the production of polypeptides having specific antigen-binding properties of Fv domains, for example, insertable variable fragments of antibodies, and modified α1-α2 domains of NKG2D ligands.

This application is a divisional of U.S. application Ser. No. 14/959,745, filed on Dec. 4, 2015 (allowed); which claims priority from U.S. Provisional Application No. 62/088,456, filed on Dec. 5, 2014 (expired).

The instant application contains a Sequence Listing which has been filed electronically in xml format and is hereby incorporated by reference in its entirety. Said xml copy, created on Sep. 13, 2022, is named SEQ LIST.xml and is 140 KB in size.

BACKGROUND OF THE INVENTION Field of the Invention

This application relates generally to the production of polypeptides having specific antigen-binding properties of Fv domains, for example, insertable variable fragments of antibodies, and modified α1-α2 domains of NKG2D ligands.

BACKGROUND INFORMATION

An antibody (Ab), FIG. 1 , also known as an immunoglobulin (Ig), in many mammals including humans is a large, Y-shape protein used by the immune system to identify and neutralize foreign objects such as bacteria and viruses (Charles Janeway (2001). Immunobiology. (5th ed.), Chapter 3. Garland Publishing. ISBN 0-8153-3642-X. (electronic full text via NCBI Bookshelf). The antibody recognizes a unique part of the foreign target, called an antigen. Each tip of the two arms of the “Y” of an antibody contains an antigen binding site, or a paratope, (a structure analogous to a lock) that is specific for one particular epitope (similarly analogous to a key) of an antigen, allowing these two structures to bind together with precision.

Using this binding mechanism, an antibody can tag a microbe or an infected cell for attack by other parts of the immune system or can neutralize its target directly, for example, by blocking a part of a microbe that is essential for its invasion and survival. The production of antibodies is the main function of the humoral, or “adaptive”, immune system. Antibodies are secreted by plasma cells. Antibodies in nature can occur in two physical forms, a soluble form that is secreted from the cell, and a membrane-bound form that is attached to the surface of a B cell via the “stem” of the Y.

Antibodies are glycoproteins belonging to the immunoglobulin superfamily and are typically made of basic structural units—each with two large heavy chains and two small light chains. There are several different types of antibody heavy chains, and several different kinds of antibodies, which are grouped into different isotypes based on which heavy chain they possess. Five different antibody isotypes are known in mammals (Market E, Papavasiliou FN (October 2003). “V(D)J recombination and the evolution of the adaptive immune system”. PLoS Biol. 1 (1): E16. doi:10.1371/journal.pbio.0000016. PMC 212695. PMID 14551913). Although the general structure of all antibodies is very similar, a small region at the tip of each arm of the Y-shaped protein is extremely variable, allowing millions of antibodies with slightly different tip structures, or antigen-binding sites, to exist. This region is known as the hypervariable or variable region. Each of these natural variants can bind to a different antigen. This enormous diversity of antibodies allows the immune system to adapt and recognize an equally wide variety of antigens (Hozumi N, Tonegawa S (1976). “Evidence for somatic rearrangement of immunoglobulin genes coding for variable and constant regions”. Proc. Natl. Acad. Sci. U.S.A. 73 (10): 3628-3632. doi:10.1073/pnas.73.10.3628. PMC 431171. PMID 824647.)

The natural “Y”-shaped Ig molecule consists of four polypeptide chains; two identical heavy chains and two identical light chains connected by disulfide bonds, FIG. 1 . Each heavy chain has two major regions, the constant region (CH) and the variable region (VH). The constant region is essentially identical in all antibodies of the same isotype, but differs in antibodies of different isotypes. A light chain also has two successive domains: a smaller constant region (CL) and the variable region (VL) (Woof J, Burton D (2004). “Human antibody-Fc receptor interactions illuminated by crystal structures.” Nat Rev Immunol 4 (2): 89-99. doi:10.1038/nri1266. PMID 15040582).

Some parts of an antibody have the same functions. Each of the two arms of the Y, for example, contains the sites that can bind to antigens and, therefore, recognize specific foreign objects. This region of the antibody is called the Fv (fragment, variable) region. It is composed of one variable domain from the heavy chain (VH) and one variable region from the light chain (VL) of the antibody(Hochman J, Inbar D, Givol D (1973). An active antibody fragment (Fv) composed of the variable portions of heavy and light chains. Biochemistry 12 (6): 1130-1135. doi:10.1021/bi00730α018. PMID 4569769). The paratope is shaped at one end of the Fv and is the region for binding to antigens. It is comprised of variable loops of (β-strands, three each on the VL and on the VH and is responsible for binding to the antigen, FIG. 2 . These 6 loops are referred to as the complementarity determining regions (CDRs) (North B, Lehmann A, Dunbrack RL (2010). “A new clustering of antibody CDR loop conformations”. J Mol Biol 406 (2): 228-256. doi:10.1016/j.jmb.2010.10.030. PMC 3065967. PMID 21035459).

Useful polypeptides that possess specific antigen binding function can be derived from the CDRs of the variable regions of antibodies. These two antibody variable domains, one of the light chain(VL) and one from the heavy chain (VH), each with 3 CDRs can be fused in tandem, in either order, using a single, short linker peptide of 10 to about 25 amino acids to create a linear single-chain variable fragment (scFv) polypeptide comprising one each of heavy and light chain variable domains, FIG. 3 (Bird, R. E., Hardman, K. D., Jacobson, J. W., Johnson, S., Kaufman, B. M., Lee, S. M., Lee, T., Pope, S. H., Riordan, G. S., and Whitlow, M. (1988) Single-chain antigen-binding proteins, Science 242, 423-426; Huston, J. S., Levinson, D, Mudgett-Hunter, M, Tai, M-S, Novotny, J, Margolies, M. N., Ridge, R., Bruccoleri, R E., Haber, E., Crea, R., and Opperman, H. (1988). Protein engineering of antibody binding sites: Recovery of specific activity in an anti-digoxin single-chain Fv analogue produced in Escherichia coli. PNAS 85: 5879-5883).

The linker is usually rich in glycine for flexibility, as well as serine, threonine, or charged amino acids for solubility, and can either connect the N-terminus of the VH with the C-terminus of the VL, or vice versa. This protein retains the specificity of the original immunoglobulin, despite removal of the constant regions and the introduction of the single linker. This format enables one ordinarily skilled in the art of recombinant DNA technology to genetically fuse the linear scFv to the N- or C-terminus of a parent protein in order to impart to the parent protein the antigen binding properties of the scFv. There are numerous other proposed or created arrangements of polyvalent and tandem scFv regions, but importantly as described below, all have at least two spatially distant termini, FIG. 4 (Le Gall, F.; Kipriyanov, S M; Moldenhauer, G; Little, M (1999). “Di-, tri- and tetrameric single chain Fv antibody fragments against human CD19: effect of valency on cell binding”. FEBS Letters 453 (1): 164-168. doi:10.1016/50014-5793(99)00713-9. PMID 10403395).

SUMMARY OF THE INVENTION

The present disclosure relates to modified α1-α2 domains of NKG2D ligands attached to polypeptides, in some embodiments antibodies or fragments of antibodies. In some aspects, the present disclosure relates to antigen-binding peptides derived from light and heavy chain antibody variable domains, which contain two linker regions and a split variable domain.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

FIG. 1 . A cartoon of a typical mammalian antibody showing its Y-shaped structure and structural components.

FIG. 2 . A cartoon of the structure of an Fv region of a natural mammalian antibody showing the 3 labeled (Complementarity Determining Regions) CDRs of the VH and the 3 unlabeled loops of the VL CDRs, which form the paratope or antigen binding site.

FIG. 3 . A cartoon of the two possible structures of a single-chain variable fragment (scFv), with the antigen binding sites including the N-termini on the left and the C-termini on the right. The single linker region, or linker peptide, in each scFv is shown as an arrow.

FIG. 4 . Polyvalent single-chain variable fragments (scFv's). Structure of divalent (top) and trivalent (bottom) scFvs, tandem (left) and di-/trimerization format (right). Note that each has 2 or more spatially distant free termini.

FIGS. 5A and 5B. Diagram of an insertable variable fragment, iFv. Diagram of an insertable variable fragment, iFv. FIG. 5A shows the structure of variable light (VL) and variable heavy (VH) domains from FGFR3-binding antibody showing the domain topology of the iFv format. Grey arrows represent the 2 linker regions (LR), one and only one of which is used traditionally to connect the termini of VL and VH to create an scFv. The LR with a dotted border connected the C-terminus of VL to the N-terminus of VH (visible behind the molecule). The LR with a solid border connected the C-terminus of VH to the N-terminus of VL. Segments of the split VL domain are labeled Nt and Ct as described in text. As a result of the creation of non-natural pair of N- and C-termini between strand 1 (51) and strand 2 (S2) the VL has been divided into an N-terminal segment (VLN) and a C-terminal segment (VLC). The 6 CDRs of VL and VH are represented as the loops at the top of the figure. FIG. 5B shows the scheme of the domain layout for inserting an iFv into loop 1 (L1) of MICA-α3 with or without a spacer region (SR). An iFv could also be similarly inserted into loop 2 (L2) and/or loop 3 (L3).

FIG. 6 . Titration curves for the modified sMICA molecules binding to FGFR3 coated wells. Bound sMICA was detected by ELISA using NKG2D-Fc to confirm the bispecific activity. Both versions of the inserted variable fragments (MICA-α3-iFv.1 and MICA-α3-iFv.2) bound FGFR3 comparably to the C-terminal fusion of an scFv (MICA-scFv).

FIGS. 7A and 7B. Thermal stability of MICA-α3-iFv.2. ELISA titration curves of MICA-scFv (FIG. 7A) or MICA-α3-iFv.2 (FIG. 7B) binding to FGFR3-coated wells after exposure to the indicated temperatures (degrees Celsius) for 1 hour. The MICA-α3-iFv exhibited strong binding to FGFR3 after exposure to 80° C., whereas MICA-scFv lost significant activity after exposure to 70° C.

FIG. 8 . NK-mediated target cell lysis assays. NKL effector cells were co-incubated with calcein-loaded, FGFR3-expressing P815 target cells at a effector:target ratio of 15:1. Increasing concentrations of a negative control MICA (sMICA) had no effect on target cell lysis, whereas the indicated FGFR3-binding MICA-α3-IFv variants stimulated target cell lysis. Compared to MICA-scFv, both MICA-α3-iFv variants directed greater target cell lysis.

FIGS. 9A and 9B. Target binding and cell lysis activity of a CD20-specific sMICA variant. MICA-α3-iFv.3 exhibited titratable binding to CD20-coated wells in an ELISA (FIG. 9A), and also enhanced NK-mediated cell lysis of CD20-expressing Ramos cells (FIG. 9B). In the experiments shown in FIG. 9B, NKL effector cells were co-incubated with calcein-loaded CD20-expressing Ramos cells at a effector:target ratio of 15:1, and increasing concentrations of either the negative control (sMICA) or MICA-α3-iFv.3.

FIG. 10 . Titration curves for the NKG2DL-α3-iFv.2 proteins binding to FGFR3-coated wells. Bound protein was detected by ELISA using NKG2D-Fc to confirm the bispecific activity. All versions of the NKG2DL-α3-iFv.2 proteins tested (OMCP, ULBP1, 2, 3, 4, 6) bound FGFR3 similarly.

FIG. 11 . NK-mediated target cell lysis assays. NKL effector cells were co-incubated with calcein-loaded, FGFR3-expressing P815 target cells at a effector:target ratio of 15:1. Increasing concentrations of a negative control MICA (sMICA) had no effect on target cell lysis, whereas each indicated NKG2DL-α3-iFv.2 protein stimulated target cell lysis.

FIGS. 12A and 12B. Structure-directed mutagenesis of the α1-α2 domain of MICA for enhanced NKG2D affinity. FIG. 12A shows the structure of the α1-α2 domain of MICA (PDB 1HYR) with the NKG2D-binding surface mapped to 57 residues colored dark grey.

FIG. 12B shows six positions that were identified as key sites for NKG2D affinity mutations. The wild-type amino acid residues are labeled and their side chains shown in dark grey spheres.

FIGS. 13A and 13B. NKG2D-Fc competition ELISAs to affinity rank α1-α2 variants. FIG. 13A shows titration data for a panel of α1-α2 affinity variants (15-18), wild-type (WT), or WED soluble MICA proteins inhibiting human NKG2D-Fc binding to plate-coated MICA. FIG. 13B shows the same set of proteins in FIG. 13A titrated against mouse NKG2D-Fc. In both assays variants 15, 16, 17, and 18 display IC₅₀ values significantly less than both WT and WED proteins. The equilibrium IC₅₀ values are shown in Table 3.

FIG. 14 . Analysis of the association and dissociation kinetics for α1-α2 variants binding to NKG2D, as measured by biolayer interferometry on an Octet instrument. Kinetic traces for a panel of α1-α2 variants. The association and dissociation phases were fit using a single exponential 1:1 binding equation and on- and off-rate constants derived from the fits are shown in Table 3.

FIG. 15 . NK-mediated target cell killing assay for the α1-α2 variants targeting FGFR3-expressing target cells. NKL effector cells were co-incubated with calcein-loaded, FGFR3-expressing P815 target cells at a effector:target ratio of 15:1. Increasing concentrations of a negative control MICA (sMICA) had no effect on target cell lysis, whereas the indicated α1-α2 variants stimulated target cell lysis. Relative to WT and WED-MICA, variants 16, 17, and 18 exhibited significantly increased killing at low concentrations.

FIG. 16 . Analysis of the association and dissociation kinetics for α1-α2 variants 20, 25, and 48 binding to NKG2D, as measured by biolayer interferometry on an Octet instrument. The association and dissociation phases were fit using a single exponential 1:1 binding equation, and on- and off-rate constants derived from the fits are shown in Table 5,

FIG. 17 . NK-mediated target cell killing, calcein-based assay for α1-α2 variants 16, 25 and WED targeting FGFR3-expressing P815 target cells.

FIG. 18 . Protein sequence alignment of α1-α2 domains from MICA and ULBPs (SEQ ID NOs.: 77-83). Amino acids highlighted in grey were selected for NNK mutagenesis in ULBP2 (60 amino acids) and ULBP3 (36 amino acids). Residues highlighted in black were identified as key positions for selected and identified as mutations that modulate binding affinity to NKG2D (Tables 6 and 7).

FIGS. 19A and 19B. Phage ELISA titrations of ULBP variants binding to NKG2D. FIG. 19A depicts experiments in which ULBP2 variants displayed on phage were titrated against NKG2D and relative binding affinities were measured relative to native ULBP2 (WT, black circles). FIG. 19B depicts experiments in which ULBP3 variants displayed on phage were titrated against NKG2D and relative binding affinities were measured relative to native ULBP3 (WT, black circles).

FIGS. 20A-D. Fusions of native (WT), modified variant WED, 25 or 48 α1-α2 domains to heavy chain (FIG. 20A) or light chain (FIG. 20B) of an FGFR3-specific antibody affected NK-dependent target cell killing. Fusions of variants 25 and 48 to either heavy chain (FIG. 20C) or light chain (FIG. 20D) significantly enhanced the extent of killing and the potency of killing compared to the WED variant and to the native (WT) fusions.

FIGS. 21A-C. Fusions of variant 25 α1-α2 domain to the heavy chains or light chains of antibodies targeting human EGFR (FIG. 21A), HER2 (FIG. 21B), or PDL1 (FIG. 21C) each enhanced NKL cell-mediated target cell killing The poor or absent killing by the respective parent antibodies, cetuximab (FIG. 21A), trastuzumab (FIG. 21B), and anti-PDL1 (FIG. 21C) are shown.

FIGS. 22A and 22B. Trastuzumab-based fusions of variant 25 α1-α2 domain arm NK cells in vivo. Parent trastuzumab, trastuzumab HC 25 fusion, and trastuzumab LC 25 fusion were conjugated with Alexa Flour. Groups of three C57BL/6 mice were injected with a single dose of 100 μg of parent, HC fusion or LC fusion; and blood was drawn from each animal at indicated times for plasma PK ELISAs (FIG. 22A) and flow cytometric analyses of the fluorescently labeled molecules bound to peripheral NK cells (FIG. 22B).

FIGS. 23A-C. Anti-drug antibodies raised in the same animals described in Example 7 and FIG. 21 administered Trastuzumab parent (FIG. 23A),Trastuzumab-based HC (FIG. 23B) and Trastuzumab-LC (FIG. 23C) fusions to variant 25. The control (Ctrl) plasma was from a mouse not administered any antibody-containing agent.

FIGS. 24A and 24B. Antibodies generated in animals administered variant 25 α1-α2 domain fusions to trastuzumab-HC and —LC, as described in Example 7 and FIGS. 21-22 , bound to both the parent antibody (FIG. 24A) and to the α1-α2 domain (FIG. 24B).

FIG. 25 . Anti-tumor activity of an anti-PDL1 fusion to variant 25. Syngeneic MC38 tumors were implanted subcutaneously in C57BL/6 mice, and tumors grew to an average of 100 mm³ before the initiation of treatment. Upon initiation of treatment four cohorts of 10 mice per group were treated parenterally with vehicle, anti-CTLA4 (100 ug i.p.), parent anti-PDL1 (300 ug i.v.), or anti-PDL1 HC_25 fusion (300 ug i.v.) on days 1, 4, and 7. Tumor volumes (cubic mm) were measured in each animal at the indicated times.

DETAILED DESCRIPTION OF THE INVENTION

In some aspects, the present invention relates to insertable variable fragment (iFv) peptides. Because the C-terminus and N-terminus of scFv molecules including polyvalent scFv structures are far apart spatially, scFv structures cannot be inserted into a loop region embedded within a protein fold of a parent or recipient protein without disrupting or destabilizing its fold(s) and/or without disrupting the Fv framework required to properly position the CDRs or hypervariable regions to retain their antigen-binding properties.

To insert the variable fragment of an antibody containing up to 6 CDRs into one or more loop regions of a nascent parent protein molecule without disrupting structural folds of the variable fragment or of the parent protein, we invented a new class of antigen-binding peptides derived from the light and heavy chain antibody variable domains. The new structures contained two linker regions, rather than the traditional single linker of scFv structures, plus a split variable domain. Conceptually the canonical termini of the variable light (VL) and heavy (VH) domains were fused into a continuous or “circular” peptide. That circular peptide structure containing all 6 CDRs of the Fv can then conceptually be split at one of several possible novel sites to create an insertable Fv (iFv). The non-natural split site can be created within either the light or the heavy chain variable domain at or near the apex or turn of a loop to create new, unique N- and C-termini spatially positioned proximal to each other, preferably within 0.5 to 1.5 nm, so as to be insertable into loops of other (parent or recipient) proteins or polypeptides without disrupting the structure, stability, or desirable function. This new class of peptides is called an insertable variable fragment (iFv). The binding or targeting specificity conveyed by an iFv to a recipient molecule can be changed by inserting into the recipient another or different iFV based on a different antibody or scFv or by replacing 1 or more of the CDRs of an existing insertable iFv.

The insertion of one or more iFv polypeptides exhibiting specific antigen-binding properties of Fv domains into other proteins and thereby imparting novel binding properties will have multiple utilities. Such uses include but are not limited to enabling the parent protein to bind the specific antigen, target the antigen, detect the presence of antigen, remove the antigen, contact or draw near the antigen, to deliver a payload to the antigen or antigen-expressing cell, recruit the antigen, and image the presence of the antigen. A payload could be conjugated directly to one or both the amino-terminus and carboxy-terminus of an iFv or indirectly to an iFv via a parent protein or peptide. Examples of payloads include but are not limited to a chromophore, a fluorophore, a pharmacophore, an atom, a heavy or radioactive isotope, an imaging agent, a chemotherapeutic agent, or a toxin. A payloaded iFv can be used to locate or identify the presence of a target molecule to which the iFv specifically binds and as such can serve as in vitro or in vivo imaging agents or diagnostic agents that are small and stable. In addition, to one or both the amino-terminus and carboxy-terminus of an iFv peptide a chemotherapeutic agent or toxic molecule can be conjugated in order to create an iFv-drug conjugate, for example, as treatment for a malignancy or infection. A single payload may be conjugated to both the amino-terminus and the carboxy-terminus of an iFv peptide so as to span or connect the two termini; such spanning may further stabilize the iFv by blocking the termini from exopeptidase degradation or protecting the iFv from denaturation or unfolding.

Examples of parent or recipient proteins or polypeptides that are candidates for insertions of iFv peptides include but are not limited to antibodies, proteins comprised of Ig folds or Ig domains, globulins, albumens, fibronectins and fibronectin domains, integrins, fluorescent proteins, enzymes, outer membrane proteins, receptor proteins, T-cell receptors, chimeric antigen receptors, viral antigens, virus capsids, viral ligands for cell receptors, high molecular weight bacteriocins, histones, hormones, knottins, cyclic peptides or polypeptides, major histocompatibility (MHC) family proteins, MIC proteins, lectins, and ligands for lectins. It is also possible to insert iFv structures into non-protein recipient molecules such a polysaccharides, dendrimers, polyglycols, peptidoglycans, antibiotics, and polyketides.

Natural killer (NK) cells and certain (CD8+ αβ and γδ) T-cells of the immunity system have important roles in humans and other mammals as first-line, innate defense against neoplastic and virus-infected cells (Cerwenka, A., and L.L. Lanier. 2001. NK cells, viruses and cancer. Nat. Rev. Immunol. 1:41-49). NK cells and certain T-cells exhibit on their surfaces NKG2D, a prominent, homodimeric, surface immunoreceptor responsible for recognizing a target cell and activating the innate defense against the pathologic cell (Lanier, LL, 1998. NK cell receptors. Ann. Rev. Immunol. 16: 359-393; Houchins JP et al. 1991. DNA sequence analysis of NKG2, a family of related cDNA clones encoding type II integral membrane proteins on human NK cells. J. Exp. Med. 173: 1017-1020; Bauer, S et al., 1999. Activation of NK cells and T cells by NKG2D, a receptor for stress-inducible MICA. Science 285: 727-730). The human NKG2D molecule possesses a C-type lectin-like extracellular domain that binds to its cognate ligands, the 84% sequence identical or homologous, monomeric MICA and MICB, polymorphic analogs of the Major Histocompatibility Complex (MHC) Class I chain-related glycoproteins (MIC) (Weis et al. 1998. The C-type lectin superfamily of the immune system. Immunol. Rev. 163: 19-34; Bahram et al. 1994. A second lineage of mammalian MHC class I genes. PNAS 91:6259-6263; Bahram et al. 1996a. Nucleotide sequence of the human MHC class I MICA gene. Immunogenetics 44: 80-81; Bahram and Spies TA. 1996. Nucleotide sequence of human MHC class I MICB cDNA. Immunogenetics 43: 230-233). Non-pathologic expression of MICA and MICB is restricted to intestinal epithelium, keratinocytes, endothelial cells and monocytes, but aberrant surface expression of these MIC proteins occurs in response to many types of cellular stress such as proliferation, oxidation and heat shock and marks the cell as pathologic (Groh et al. 1996. Cell stress-regulated human MHC class I gene expressed in GI epithelium. PNAS 93: 12445-12450; Groh et al. 1998. Recognition of stress-induced MHC molecules by intestinal γδT cells. Science 279: 1737-1740; Zwirner et al. 1999. Differential expression of MICA by endothelial cells, fibroblasts, keratinocytes and monocytes. Human Immunol. 60: 323-330). Pathologic expression of MIC proteins also seems involved in some autoimmune diseases (Ravetch, J V and Lanier L L. 2000. Immune Inhibitory Receptors. Science 290: 84-89; Burgess, S J. 2008. Immunol. Res. 40: 18-34). The differential regulation of NKG2D ligands, such as the polymorphic MICA and MICB, is important to provide the immunity system with a means to identify and respond to a broad range of emergency cues while still protecting healthy cells from unwanted attack (Stephens H A, (2001) MICA and MICB genes: can the enigma of their polymorphism be resolved? Trends Immunol. 22: 378-85; Spies, T. 2008. Regulation of NKG2D ligands: a purposeful but delicate affair. Nature Immunol. 9: 1013-1015).

Viral infection is a common inducer of MIC protein expression and identifies the viral-infected cell for NK or T-cell attack (Groh et al. 1998; Groh et al. 2001. Co-stimulation of CD8+ αβT-cells by NKG2D via engagement by MIC induced on virus-infected cells. Nat. Immunol. 2: 255-260; Cerwenka, A., and L. L. Lanier. 2001). In fact, to avoid such an attack on its host cell, cytomegalovirus and other viruses have evolved mechanisms that prevent the expression of MIC proteins on the surface of the cell they infect in order to escape the wrath of the innate immunity system (Lodoen, M., K. Ogasawara, J.A. Hamerman, H. Arase, J.P. Houchins, E.S. Mocarski, and L.L. Lanier. 2003. NKG2D-mediated NK cell protection against cytomegalovirus is impaired by gp40 modulation of RAE-1 molecules. J. Exp. Med. 197:1245-1253; Stern-Ginossar et al., (2007) Host immune system gene targeting by viral miRNA. Science 317: 376-381; Stern-Ginossar et al., (2008) Human microRNAs regulate stress-induced immune responses mediated by the receptor NKG2D. Nature Immunology 9: 1065-73; Slavuljica, I A Busche, M Babic , M Mitrovic, I Gas̆parovic, D Cekinovic, E Markova Car, EP Pugel, A Cikovic, VJ Lisnic, WJ Britt, U Koszinowski, M Messerle, A Krmpotic and S Jonjic. 2010. Recombinant mouse cytomegalovirus expressing a ligand for the NKG2D receptor is attenuated and has improved vaccine properties. J. Clin. Invest. 120: 4532-4545).

In spite of their stress, many malignant cells, such as those of lung cancer and glioblastoma brain cancer, also avoid the expression of MIC proteins and as a result may be particularly aggressive as they too escape the innate immunity system (Busche, A et al. 2006, NK cell mediated rejection of experimental human lung cancer by genetic over expression of MEW class I chain-related gene A. Human Gene Therapy 17: 135-146; Doubrovina, E S, M M Doubrovin, E Vider, R B Sisson, R J O'Reilly, B Dupont, and Y M Vyas, 2003. Evasion from N K Cell Immunity by MHC Class I Chain-Related Molecules Expressing Colon Adenocarcinoma (2003) J. Immunology 6891-99; Friese, M. et al. 2003. MICA/NKG2D-mediated immunogene therapy of experimental gliomas. Cancer Research 63: 8996-9006; Fuertes, M B, M V Girart, L L Molinero, C I Domaica, L E Rossi, M M Barrio, J Mordoh, G A Rabinovich and N W Zwirner. (2008) Intracellular Retention of the NKG2D Ligand MHC Class I Chain-Related Gene A in Human Melanomas Confers Immune Privilege and Prevents NK Cell-Mediated Cytotoxicity. J. Immunology, 180: 4606 -4614).

The high resolution structure of human MICA bound to NKG2D has been solved and demonstrates that the α3 domain of MICA has no direct interaction with the NKG2D (Li et al. 2001. Complex structure of the activating immunoreceptor NKG2D and its MHC class I-like ligand MICA. Nature Immunol. 2: 443-451; Protein Data Bank accession code 1HYR). The α3 domain of MICA, like that of MICB, is connected to the α1-α2 platform domain by a short, flexible linker peptide, and itself is positioned naturally as “spacer” between the platform and the surface of the MIC expressing cell. The 3-dimensional structures of the human MICA and MICB α3 domains are nearly identical (root-mean square distance <1 A on 94 C-aa's) and functionally interchangeable (Holmes et al. 2001. Structural Studies of Allelic Diversity of the MHC Class I Homolog MICB, a Stress-Inducible Ligand for the Activating Immunoreceptor NKG2D. J Immunol. 169: 1395-1400).

As used herein, a “soluble MIC protein”, “soluble MICA” and “soluble MICB” refer to a MIC protein containing the α1, α2, and α3 domains of the MIC protein but without the transmembrane or intracellular domains.

The α1-α2 platform domain of a soluble MIC protein is tethered to the α3 domain and is diffusible in the intercellular or intravascular space of the mammal. Preferably the α1-α2 platform domains of the non-natural MIC proteins of the invention are at least 80% identical or homologous to a native or natural α1-α2 domain of a human MICA or MICB protein and bind NKG2D. In some embodiments, the α1-α2 platform domain is 85% identical to a native or natural α1-α2 platform domain of a human MICA or MICB protein and binds NKG2D. In other embodiments, the α1-α2 platform domain is 90%, 95%, 96%, 97%, 98%, or 99% identical to a native or natural α1-α2 platform domain of a human MICA or MICB protein and binds NKG2D.

In some embodiments, a heterologous peptide tag may be fused to the N-terminus or C-terminus of an α1-α2 domain or a soluble MIC protein to aid in the purification of the soluble MIC protein. Tag sequences include peptides such as a poly-histidine, myc-peptide or a FLAG tag. Such tags may be removed after isolation of the MIC molecule by methods known to one skilled in the art.

As used herein “peptide”, “polypeptide”, and “protein” are used interchangeably; and a “heterologous molecule”, “heterologous peptide”, “heterologous sequence” or “heterologous atom” is a molecule, peptide, nucleic acid or amino acid sequence, or atom, respectively, that is not naturally or normally found in physical conjunction with the subject molecule.

The term “comprising,” which is used interchangeably with “including,” “containing,” or “characterized by,” is inclusive or open-ended language and does not exclude additional, unrecited elements or method steps. The phrase “consisting of” excludes any element, step, or ingredient not specified in the claim. The phrase “consisting essentially of” limits the scope of a claim to the specified materials or steps and those that do not materially affect the basic and novel characteristics of the claimed invention. The present disclosure contemplates embodiments of the invention compositions and methods corresponding to the scope of each of these phrases. Thus, a composition or method comprising recited elements or steps contemplates particular embodiments in which the composition or method consists essentially of or consists of those elements or steps.

All references cited herein are hereby incorporated by reference in their entireties, whether previously specifically incorporated or not. As used herein, the terms “a”, “an”, and “any” are each intended to include both the singular and plural forms.

Having now fully described the invention, it will be appreciated by those skilled in the art that the same can be performed within a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While this invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications. This application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and may be applied to the essential features hereinbefore set forth.

EXAMPLES of iFv and of Modified al- α2 Domains of NKG2D Ligands

Example 1 (iFv). As specific examples, we synthesized a 1126 bp and a 1144 bp DNA fragment (SEQ ID NO:1 and 2, respectively) encoding in the following order: the α3 domain of human MICA (as a parent peptide) amino acid 182 to amino acid 194 (the beginning of loop 1 of the α3 domain), no spacer or a GGS amino acid spacer region (SR), an iFv peptide based on the structure of a Fibroblast Growth Factor Receptor 3 (FGFR3)-binding antibody (MAbR3;Qing, J., Du, X., Chen, Y., Chan, P., Li, H., Wu, P., Marsters, S., Stawicki, S., Tien, J., Totpal, K., Ross, S., Stinson, S., Dornan, D., French, D., Wang, Q. R., Stephan, J. P., Wu, Y., Wiesmann, C., and Ashkenazi, A. (2009) Antibody-based targeting of FGFR3 in bladder carcinoma and t(4;14)-positive multiple myeloma in mice, The Journal of clinical investigation 119, 1216-1229.), no spacer or another GGS spacer region, the distal portion of loop 1 of the α3 domain starting at amino acid 196 and including the remaining carboxy-terminal portion of the α3 domain to amino acid 276 of a soluble MICA molecule. Each synthetic, double stranded DNA polynucleotide then encoded a polypeptide that contained 6 CDRs in the form of an iFv inserted into loop 1 of the α3 domain of MICA.

This iFv peptide itself (SEQ ID NO.:3), encoded by SEQ ID NO.:4, contained two identical, typical linker regions (LR) corresponding to residues GGSSRSSSSGGGGSGGGG (SEQ ID NO.:5) (Andris-Widhopf, J., Steinberger, P., Fuller, R., Rader, C., and Barbas, C. F., 3rd. (2011) Generation of human Fab antibody libraries: PCR amplification and assembly of light- and heavy-chain coding sequences, Cold Spring Harbor protocols 2011). One LR joined the C-terminus of VL to the N-terminus of the VH domain, and the second LR joined the C-terminus of the VH domain to the N-terminus of VL. Conceptually this new structure is the continuous or “circular” peptide referred to above and contained 6 CDRs of the starting Fv. The variable VL chain of the antibody was effectively split within the loop region between beta-strands 1 and 2 (51 and S2) and thereby created a new N-terminal segment (VLN) and a new C-terminal segment (VLC) with an accompanying pair of new, non-natural C- and N-termini, respectively, FIG. 5A. This pair of termini created a sole site for attachment or conjugation of the iFv to the recipient molecule such as a protein. The schematic of the inserted iFv in the parent α3 domain is shown in FIG. 5B.

To produce the soluble MICA proteins with a heterologous iFv peptide inserted into the α3 domain we generated a baculoviral expression vector to accommodate the DNA sequences (SEQ ID NO.s:1 and 2) encoding the α3-iFv.1 (SEQ ID NO.:6) and α3-iFv.2 (SEQ ID NO.:7), respectively. The DNA fragments were amplified by PCR, digested using Ncol and EcoRI restriction enzymes, and subcloned into the baculoviral expression vector, SW403, replacing the wild-type α3 domain. SW403 is a baculoviral expression vector derived from pVL1393 (Invitrogen, Inc.) into which wild-type sMICA (residues 1-276) had previously been cloned using 5′ BamHI and 3′ EcoRI sites. The new expression vector was co-transfected with baculoviral DNA into SF9 insect cells, and baculovirus was grown for two amplification cycles and used to express the His-tagged MICA-α3-IFv proteins in T.ni insect cells according to manufacturer's protocol (Invitrogen). The expression was carried out in a 100 mL volume for three days and the growth medium was harvested for purification of the secreted soluble protein using Ni-affinity chromatography. Monomeric MICA-α3-IFv was purified to >90% purity with the expected molecular weight of 60.9 kDa as determined by SDS-PAGE. Functional characterization was carried out using binding ELISAs and in vitro target cell killing assays.

The purified MICA-α3-IFv proteins were tested in a FGFR3-binding ELISA to confirm simultaneous binding to the FGFR3 target and the NKG2D receptor. FGFR3 in phosphate buffered saline (PBS) was coated onto Maxisorp plates at 2 ug/ml concentration. Each MICA protein was titrated, allowed to bind FGFR3 for 1 hour, and washed to remove unbound sMICA protein. Bound MICA-α3-IFv protein was detected using NKG2D-Fc and anti-Fc-HRP conjugate. FIG. 6 shows that the binding of both MICA-α3-iFv.1 and MICA-α3-iFv.2 to FGFR3 was comparable to that of a MICA-scFv, made by fusing to the C-terminus of soluble MICA a traditional scFv constructed from MAbR3. These ELISA results also indicated that both the FGFR3 and NKG2D binding specificities of the scFv and the α1-α2 domain, respectively, were retained by the modified MICA and demonstrated that the iFv peptide inserted using different spacer formats was functional.

We tested and compared the thermal stability of sMICA-α3-iFv.2 to that of sMICA-scFv. Both proteins were subjected for 1 hr to increasing temperatures from 60-90 ° C. and then allowed to equilibrate to room temperature for 1 hour before being assayed for binding properties by ELISA. The results in FIGS. 7A and 7B showed that MICA-α3-iFv.2 can be subjected to temperatures as high as 80° C. with no loss in specific binding to FGFR3. The traditional MICA-scFv lost binding activity at 70° C. This result indicated that soluble MICA containing the invented iFv format is significantly more stable than terminal fusions of a traditional scFv (Miller, B. R., Demarest, S. J., Lugovskoy, A., Huang, F., Wu, X., Snyder, W. B., Croner, L. J., Wang, N., Amatucci, A., Michaelson, J. S., and Glaser, S. M. (2010) Stability engineering of scFvs for the development of bispecific and multivalent antibodies, Protein engineering, design & selection : PEDS 23, 549-557; Weatherill, E. E., Cain, K. L., Heywood, S. P., Compson, J. E., Heads, J. T., Adams, R., and Humphreys, D. P. (2012) Towards a universal disulphide stabilised single chain Fv format: importance of interchain disulphide bond location and vL-vH orientation, Protein engineering, design & selection : PEDS 25, 321-329).

The ability of MICA-α3-iFv to redirect NK cell-mediated lysis of FGFR3-expressing target cells was demonstrated in vitro in a calcein-release assay. The Natural Killer (NK) cell line, NKL, was co-cultured with calcein-loaded P815 target cells ectopically expressing FGFR3. The results in FIG. 8 showed that the two MICA-α3-iFv molecules induced significantly greater NK-mediated lysis compared to the traditional MICA-scFv fusion, while the non-targeted soluble MICA control had no killing activity. These results confirmed that the invented iFv bound FGFR3 on target cells and in the context of the complete parent protein molecule, soluble MICA, induced potent NK cell-mediated lysis.

The applicability of the iFv format to other antibody variable domains was demonstrated by similarly constructing an α3-iFv.3 (SEQ ID NO.:8), which contained an iFv derived from a CD20-specific antibody (Du, J., Wang, H., Zhong, C., Peng, B., Zhang, M., Li, B., Huo, S., Guo, Y., and Ding, J. (2007) Structural basis for recognition of CD20 by therapeutic antibody Rituximab, The Journal of biological chemistry 282, 15073-15080). FIGS. 9A and 9B show that MICA-α3-iFv.3 was able to specifically bind wells coated with CD20 in a plate-based

ELISA as described above and also induced NK-mediated lysis of Ramos cells expressing CD20 in a calcein-release assay.

Example 2 (Modified al- α2 Domains of NKG2D Ligands). Human proteins designated ULBP-1 through ULBP-6 are, like MICA and MICB, naturally occurring, stress-induced, cell surface ligands that bind NKG2D receptors on and activate human NK cells and certain T-cells (15; Cerwenka A, Lanier LL (2004). NKG2D ligands: unconventional MHC class I-like molecules exploited by viruses and cancer. Tissue Antigens 61 (5): 335-43. doi:10.1034/j.1399-0039.2003.00070.x. PMID 12753652). In addition, the cowpox virus protein OMCP is a secreted domain that like the α1-α2 domain of MIC proteins binds NKG2D. OMCP exhibits a very high affinity for NKG2D, apparently in order to block NKG2D′s recognition of the natural stress ligands induced by the virus on its infected host cell (Eric Lazear, Lance W. Peterson, Chris A. Nelson, David H. Fremont. J Virol. 2013 January; 87(2): 840-850. doi: 10.1128/JVI.01948-12). While the ULBPs and OMCP are considered NKG2D ligands (NKG2DLs) that share the canonical α1-α2 domain structure, the sequence homology with MICA α1-α2 is less than 27%, and they all naturally lack an α3 domain for tethering targeting domains. We constructed a series of non-natural ULB and OMCP proteins by attaching the heterologous polypeptides that specifically targeted and killed FGFR3-expressing cells as the result of fusing to each of ULBP-1, ULBP-2, ULBP-3, ULBP-4, ULBP-6 and OMCP, a modified α3 domain of MICA into which a targeting iFv had been inserted. In addition, we modified the α1-α2 domain of MICA to enhance the affinity of α1-α2 domain for NKG2D and then attached to the modified α1-α2 domains heterologous molecules such as polypeptides. To produce the proteins consisting of ULBP and OMCP α1-α2 domains attached to modified α3-IFv domains we generated a baculoviral expression vector to accommodate the DNA fragments

(SEQ ID NOs:9-14) that encoded the different α1-α2 domains of ULBP-1, ULBP-2, ULBP-3, ULBP-4, ULBP-6, and OMCP (SEQ ID NOs:15-20, respectively). The DNA fragments were amplified by PCR, digested using BlpI and Ncol restriction enzymes, and individually subcloned into the baculoviral expression vector, KLM44, replacing the MICA α1-α2 domain. KLM44 was a baculoviral expression vector derived from SW403 into which MICA-α3-iFv.2 had previously been cloned (example 1). The new NKG2DL-α3-iFv.2 constructs, containing the ULBPs and OMCP α1-α2 domain fusions to α3-iFv.2 (ULBP1-α3-iFv.2, ULBP2-α3-iFv.2, ULBP3-α3-iFv.2, ULBP4-α3-iFv.2, ULBP6-α3-iFv.2, and OMCP-α3-iFv.2; SEQ ID NO.:21-26, respectively), were co-transfected with baculoviral DNA into SF9 insect cells. Baculovirus was grown for two amplification cycles and used to express these His-tagged NKG2DL-α3-iFv.2 proteins in T.ni insect cells according to manufacturer's protocol (Invitrogen). The expression was carried out in a 100 mL volume for three days and the growth medium was harvested for purification of the secreted soluble protein using Ni-affinity chromatography. Monomeric proteins of correct molecular weight were purified to >90% purity as determined by SDS-PAGE. Functional characterization was carried out using binding ELISAs and in vitro target cell killing assays.

The 6 purified NKG2DL-α3-iFv.2 proteins were tested in a FGFR3-binding ELISA to confirm simultaneous binding to the FGFR3 target and the NKG2D receptor. FGFR3 in phosphate buffered saline (PBS) was coated onto Maxisorp plates at 2 ug/ml concentration. Each NKG2DL-α3-iFv.2 protein was titrated, allowed to bind FGFR3 for 1 hour, and washed to remove unbound protein. The bound NKG2DL-α3-iFv.2 protein was detected using NKG2D-Fc and anti-Fc-HRP conjugate. FIG. 10 shows that all 6 NKG2DL-α3-iFv.2 proteins bound potently to FGFR3, as expected, through interaction with the iFv.2 domain, and the NKG2D binding activity was retained by the attached NKG2DL α1-α2 domains, which demonstrated that the attached α3-IFv domain imparted functional FGFR3 binding activity to the ULBP and OMPC proteins that, like MIC proteins, bind NKG2D.

The ability of the NKG2DL-α3-iFv.2 proteins to redirect NK cell-mediated lysis of FGFR3-expressing target cells was demonstrated in vitro in a calcein-release assay. The Natural Killer (NK) cell line, NKL, was co-cultured with calcein-loaded P815 target cells ectopically expressing FGFR3. The results in FIG. 11 showed that OMCP-α3-iFv.2 induced the greatest NK-mediated lysis, while the other NKG2DL-α3-iFv.2 proteins all displayed specific killing activity with varying degrees of potency and amount of lysis. These results confirmed that the invented iFv imparts specific binding activity to other proteins that retained their own functional properties and induced different levels of cell-mediated lysis of iFv-targeted cells.

Example 3 (Modified α1-α2 Domains of NKG2D Ligands). These are examples of attaching polypeptides to NKG2DLs which were modified to significantly enhance their binding affinity to the human NKG2D receptor. The α1-α2 domain of MIC proteins is an NKG2DL for the NKG2D receptor. This affinity is sufficient for physiologic activation of NK cells and stimulating lysis of cells expressing native full-length MIC proteins irreversibly tethered to the two-dimensional plasma membrane surface of a “target cell” (Bauer S, Groh V, Wu J, Steinle A, Phillips JH, Lanier LL, Spies T., Science. 1999 Jul 30;285(5428):727-9.). However, because engineered soluble MIC proteins of the instant invention reversibly bind specific target antigens on the surface of a target cell, the binding affinity of the engineered soluble MIC protein to NKG2D will directly affect the stability of the soluble MIC-dependent complex formed between NK cells and cells expressing target antigens. Especially if the affinity between sMICA and NKG2D is increased by a substantially slower dissociation rate or off-rate of the modified sMICA from NKG2D, the NK cell-based killing would be expected to be greater at lower densities of soluble MIC molecules bound to a target cell. Prior to the instant invention there had not been identified any α1-α2 mutations that alter the killing activity of soluble MIC proteins or significantly reduce the binding off-rate to enhance affinity of MIC proteins to NKG2D. A computational design effort showed that three mutations in the α1-α2 domain of wild-type MICA: N69W, K152E, and K154D (WED-MICA) in combination can moderately affect NKG2D binding affinity by affecting the stability of unbound MICA and thereby its association rate or on-rate of binding to NKG2D (Lengyel CS, Willis LJ, Mann P, Baker D, Kortemme T, Strong RK, McFarland BJ.J Biol Chem. 2007 Oct 19;282(42):30658-66. Epub 2007 Aug 8); Subsequent extensive computational design work by the same group scanning by iterative calculations 22 amino acid positions of MICA theoretically in contact with NKG2D, according to the published structural descriptions (Li P, Morris DL, Willcox BE, Steinle A, Spies T, Strong RK., Nat Immunol. 2001 May;2(5):443-451), showed experimentally that when combined with the earlier designed 3 changes, further rational, iterative computational design of MICA qualitatively changed its affinity for NKG2D from weak (Kd ˜2.5 μM) to moderately tight (Kd 51 nM) ⁻with a total of seven combined mutations (1-lenager, Samuel Melissa A. Hale, Nicholas J. Maurice, Erin C. Dunnington, Carter J. Swanson, Megan J. Peterson, Joseph J. Ban, David. J. Culpepper, Luke D. Davies, Lisa K. Sanders, and Benjamin J. McFarland, 2102, Combining different design strategies for rational affinity maturation of the MICA-NKG2D interface. Protein Science 21:1396-1407). In contrast, the experimental approach described in the instant invention experimentally selected amino acid modifications of MICA that slowed the off-rate between the α1-α2 domain of MICA and NKG2D, commencing with a MICA stabilized.

by the 3 WED changes of Lengyel et al (Lengyel CS, Willis L J, Mann P, Baker D, Kortemme T, Strong R K, McFarland B J., J Biol Chem. 2007 Oct. 19; 282(42):30658-66. Epub 2007 Aug 8).

This example of the instant invention relates to modifying the NKG2D binding affinity of soluble MIC proteins through engineering specific mutations at selected amino acid positions within the α1-α2 domain that influence the off-rate binding kinetics and thereby alter the NK cell-mediated killing activity of the invented non-natural, targeted MIC molecules.

To engineer soluble non-natural α1-α2 domains with altered affinity to NKG2D 57 residues in the α1-α2 domain were chosen for extensive mutagenesis (FIG. 12A). Synthetic DNA libraries coding for the α1-α2 domain and containing NNK mutagenic codons at each of the 57 amino acid positions were synthesized, individually cloned as fusions to the pIII minor coat protein of M13 phage, and phage particles displaying the mutagenized α1-α2 variants were produced in SS320 E.coli cells according to standard methodologies (Andris-Widhopf, J., Steinberger, P., Fuller, R., Rader, C., and Barbas, C. F., 3rd. (2011) Generation of human Fab antibody libraries: PCR amplification and assembly of light- and heavy-chain coding sequences, Cold Spring Harbor protocols 2011). The α1-α2 phage libraries were sorted for increased binding affinity using recombinant biotinylated NKG2D as the target antigen and cycled through iterative rounds of intentionally prolonged binding, prolonged washing, and eluting of the phage clones in order to select high affinity variants enriched for slow dissociation- or off-rates. A set of specific amino acid mutations occurred at high frequencies at 6 positions in α1-α2 and were selected as preferred amino acid substitutions with enhanced NKG2D binding affinity (FIG. 12B, Table 1).

Table 1. Selected affinity mutations at the indicated 6 amino acid positions of the α1-α2 domain of MIC. The amino acids of SEQ ID NOs.: 35 at each of the 6 positions are shown in bold in the first row of the table. The identified affinity mutations are listed in decreasing frequency from top to bottom. All amino acids are represented by the single letter IUPAC abbreviations.

S20 G68 K125 E152 H161 Q166 P L L T R F T F R V S S D S F G A H A A T F K Y L Y A Y G W N I N A L V E V Q F L T Y D Y M W I I S N S H M P

We synthesized DNA polynucleotides (SEQ ID NOs. 27-30) encoding the α1-α2 domains of 4 representative variants 15, 16, 17, 18 that contained different combinations of specific discovered mutations (Table 2).

Table 2. Sequences of specific α1-α2 domain variants. The specific amino acid substitutions for variants 15, 16, 17, and 18 (SEQ ID NOS.: 31-34, respectively) are listed relative to the amino acids of SEQ ID NO.:35 in bold. All amino acids are represented by the single letter IUPAC abbreviations.

Variant SEQ ID NO.: S20 G68 K125 H161 15 31 S G N R 16 32 S G L R 17 33 S L L R 18 34 p L L R

To the NKG2DLs in the above example, we directly attached heterologous molecules such as a polypeptide to each of these 4 modified α1-α2 NKG2DLs using a linker peptide. Four His-tagged proteins (SEQ ID NOs.: 31-34) consisting of modified NKG2DLs with attached heterologous molecules were expressed in insect cells and purified to characterize their NKG2D binding affinities and kinetic binding parameters. Using a competitive binding ELISA, we determined the relative NKG2D binding affinities of the 4 modified α1-α2 variants. A soluble wild type (WT) NKG2DL, sMICA protein, was coated in all wells of a maxisorp ELISA plate to provide a binding partner for the human NKG2D-Fc reagent. Solutions of the four α1-α2 variants as well as WT and WED- α1-α2 domains (SEQ ID NO.: 35) were titrated in the ELISA wells and allowed to competitively inhibit 2nM human NKG2D-Fc binding to the WT sMICA coated on the plate. The level of human NKG2D-Fc that bound to the WT NKG2DL on the plate was detected using an anti-Fc-HRP antibody. FIG. 13A shows variants 16, 17, and 18 exhibited IC₅₀ values of 0.7, 0.6, 0.5 nM while variant 15 exhibited an IC₅₀ value of 1.7 nM, all possessing significantly better binding to NKG2D, 27, 32-, 38- and 11-fold better, than WT NKG2DL, respectively, as well as substantially better than WED-MICA (Table 3).

Table 3. Equilibrium and kinetic binding parameters for α1-α2 variants. IC₅₀ values were derived from 4-parameter fits to the competition binding titrations (FIGS. 12A and 12B) and the kinetic binding parameters were derived from single exponential fits to the binding kinetics (FIGS. 13A and 13B). Equilibrium binding constants (K_(d)) were derived from the kinetic binding parameters using the equation K_(d)=k_(OFF)/k_(ON).

Kinetic Binding Parameters α1-α2 Variant IC₅₀ (nM) K_(ON) (M⁻¹ s⁻¹) K_(OFF) (s⁻¹) K_(d) (nM) WT 19.4 1.3 × 10⁵ 1.8 × 10⁻³ 13.8 WED 4.4 2.9 × 10⁵ 1.7 × 10⁻³ 5.9 15 1.7 0.7 × 10⁵ 1.1 × 10⁻⁴ 1.5 16 0.7 2.0 × 10⁵ 0.9 × 10⁻⁴ 0.5 17 0.6 2.0 × 10⁵ 0.7 × 10⁻⁴ 0.4 18 0.5 2.3 × 10⁵ 0.9 × 10⁻⁴ 0.4

Importantly, the relative IC₅₀ differences also translated to better binding to murine NKG2D-Fc (FIG. 13B), and demonstrated the ability to improve binding of soluble, modified α1-α2 domains across human and non-human NKG2D receptors, an important property for preclinical drug development.

In order to understand the kinetic basis for the altered affinities, both the on-rates and off-rates for the α1-α2 variant NKG2DLs binding to surface coated biotinylated human NKG2D were measured using biolayer interferometry (Octet) at 100 nM of each of the modified α1-α2 proteins. Consistent with results from the IC₅₀ ELISAs, variants 16, 17 and 18 each displayed significant reductions in the off-rate (18-fold relative to WT), which is largely responsible for the affinity increase (-30-fold relative to WT α1-α2)(FIG. 14 ; Table 3). Although variant 15 displayed a similar slow off-rate as did 16, 17, and 18, its on-rate was decreased, resulting in an affinity stronger than WT but weaker variants 16, 17 and 18. Because the only difference between variant 15 (SEQ ID NO.:31) and 16 (SEQ ID NO.:32) was K125N versus K125L, the mutation at position 125 clearly altered the on-rate while the decreased off-rate was attributed to the H161R mutation. Therefore, while the selected set of NKG2DL mutations (Table 1) was used to increase the α1-α2 affinity for NKG2D through significant off-rate reduction, certain substitutions also altered the on-rate resulting in a range of incremental affinity increases that we showed in this invention to have differential activity in the NK cell-mediated killing assays as described below.

The ability of the α1-α2 affinity variants to redirect NK cell-mediated lysis of FGFR3-expressing target cells was demonstrated in vitro in a calcein-release assay. The human Natural Killer (NK) cell line, NKL, was co-cultured with calcein-loaded P815 target cells ectopically expressing FGFR3 and titrated with soluble modified MIC proteins. The results in FIG. 15 showed that the killing activities of the FGFR3-specific soluble MIC variants correlated with their engineered α1-α2 affinities. Specifically, variants 16, 17, and 18 exhibited ˜15-fold more killing than WT at 0.78 nM. The WED-MICA (SEQ ID NO.:35) was only slightly better than WT. Therefore, the invention describes amino acid substitutions within the α1-α2 domain that increased the NKG2D binding affinity by reducing the off-rate of soluble MIC protein binding to human NKG2D and consequentially led to the predictably increased killing potency. WED-MICA, which exhibited somewhat greater affinity than WT MICA to NKG2D (FIG. 13A) by increasing on-rate rather than reducing off-rate (FIG. 14 ), did not exhibit substantial improvement of target cell killing (FIG. 15 ). Furthermore, as shown in FIG. 13B, WED-MICA exhibited substantially poorer binding to murine NKG2D than even WT MICA, while variants 15, 16, 17, and 18 each exhibited greater affinity for both human and murine NKG2D, FIGS. 13A and 13B.

These α1-α2 NKG2DL affinity variants 15, 16, 17, and 18 enhanced the binding affinity of the attached polypeptide to the NKG2D receptor and thereby enhanced NK cell-mediated lysis of targeted cells, FIG. 15 .

Example 4 (Modified α1-α2 Domains of NKG2D Ligands). This embodiment of the instant invention relates to additional α1-α2 NKG2DL affinity variants derived through engineering specific mutations at selected amino acid positions within the α1-α2 domain of soluble MIC molecules, as described in Example 3 (Table 1), that also influence the off-rate binding kinetics and thereby alter the NK cell-mediated killing activity of the non-natural α1-α2 domains. While variants 15-18 focused on specific mutations found at positions S20, G68, K125, and H161, another set of variants were isolated with additional mutations at E152, H158, and Q166 (Table 4).

Table 4. Sequences of specific α1-α2 domain variants. The specific amino acid substitutions for variants 20, 25, and 48 are listed relative to the amino acids of SEQ ID NO.:35, shown in bold in the first row of the table. All amino acids are represented by the single letter IUPAC abbreviations.

SEQ Variant ID NO.: S20 G68 K125 E152 H158 H161 Q166 20 39 S A L Q R H F 25 40 S G L E H R S 48 41 S G L A I R A

DNA polynucleotides (SEQ ID NOs. 36-38) encoding the α1-α2 domains of 3 representative variants 20, 25, 48 (SEQ ID NOs. 39-41, respectively) that contained different combinations of specific discovered mutations (Table 4), were synthesized. To the NKG2DLs in the above example, heterologous molecules, such as an FGFR3-binding polypeptide, were directly attached to each of these 3 modified α1-α2 NKG2DLs using a linker peptide. The constructs were cloned into the Xbal and BamHI sites of pD2509, a CMV-based mammalian cell expression vector. Three His-tagged proteins (SEQ ID NOs.: 39-41), consisting of modified NKG2DLs with attached heterologous molecules that bind to FGFR3, were transiently expressed in HEK293 cells using the Expi293 expression system according to the manufacturer's protocol (Life Technologies), and purified using Ni-affinity chromatography to obtain the isolated proteins for biochemical and activity-based analysis.

In order to characterize the NKG2D binding affinities, both the on-rates and off-rates for the three α1-α2 variant NKG2DLs binding to surface-coated biotinylated human NKG2D were measured using biolayer interferometry (Octet). Binding titrations were performed for each protein using a titration range of 1-100 nM, and the kinetic data were fitted to obtain on-rates, off-rates, and equilibrium binding constants.

Variant 25 (SEQ ID NO.: 40) contains only the addition of the Q166S mutation relative to variant 16 (SEQ ID NO.: 32) (Table 2), and exhibited a NKG2D binding affinity of 62 pM largely due to decreased off-rate (FIG. 16 and Table 5). This represented an 8-fold enhancement in equilibrium binding affinity due to the Q166S mutation (compare Table 3 and Table 5), and demonstrated that specific mutations at Q166 influenced binding affinity through decreased off-rate.

Table 5. Kinetic binding parameters for α1-α2 variants. Kinetic binding parameters were derived from single exponential fits to the binding kinetics (FIG. 16 ). Equilibrium binding constants (K_(d)) were derived from the kinetic binding parameters using the equation K_(d)=k_(OFF)/k_(ON).

Kinetic Binding Parameters α1-α2 Variant K_(ON) (M⁻¹ s⁻¹) K_(OFF) (s⁻¹) K_(d) (nM) 20 3.6 x 105 3.0 x 105 0.083 25 4.7 x 105 2.9x105 0.062 48 2.0 x 105 3.0 x 1033 15

Variant 20 (SEQ ID NO.: 39) contained the specific mutations G68A, E152Q, H158R and Q166F, and maintained binding parameters similar to variant 25 (Table 5), suggesting that this unique combination of specific mutations also has improved NKG2D binding affinity due to a decreased off-rate.

Variant 48 (SEQ ID NO.: 41) contained the K125L and H161R mutations found in variant 16 (Table 2); however the addition of mutations E152A, H1581, and Q166A (Table 4) significantly increased the off-rate, resulting in a 250-fold reduction in NKG2D binding affinity (FIG. 16 and Table 5). The Q166A mutation is not one of the favored affinity enhancement mutations selected for position Q166 (Table 1) and may have contributed to the reduction in off-rate observed. These data clearly demonstrated that unique combinations of engineered, mutations selected and identified at defined positions within α1-α2 domains tuned the NKG2D binding affinity through off-rate modulation.

The non-natural α1-α2 affinity variants with attached polypeptides redirected NK cell-mediated lysis of FGFR3-expressing target cells, as demonstrated in vitro in a calcein-release assay. The human Natural Killer (NK) cell line, NKL, was co-cultured with calcein-loaded P815 target cells ectopically expressing FGFR3, and titrated with soluble modified NKG2D ligand α1-α2 proteins. The results in FIG. 17 showed that the killing potencies of the FGFR3-targeted soluble MIC variants correlated with their engineered α1-α2 affinities. Specifically, variant 25 exhibited ˜3-fold greater killing than variant 16 at 0.2 nM, representing an ˜5-fold improvement in the EC50 for cell killing. In addition, the data clearly showed preferred killing activity across representative soluble MIC variants in the order of variant 25>16>WED (FIG. 17 ).

Example 5 (Modified α1-α2 Domains of NKG2D Ligands). This embodiment relates to additional α1-α2 NKG2DL affinity variants derived through engineering the α1-α2 domains of ULBP proteins. ULBP proteins contain α1-α2 domains, which are NKG2D ligands capable of binding to the NKG2D receptor (Cerwenka A, Lanier L L (2004). NKG2D ligands: unconventional MHC class I-like molecules exploited by viruses and cancer. Tissue Antigens 61 (5): 335-43. doi:10.1034/j.1399-0039.2003.00070.x. PMID 12753652). This affinity of NKG2D binding is sufficient for physiologic activation of NK cells and stimulating lysis of cells expressing native full-length ULBP proteins naturally and irreversibly tethered to the two-dimensional plasma membrane surface of a “target cell” (Cerwenka A, Lanier LL (2004). NKG2D ligands: unconventional WIC class I-like molecules exploited by viruses and cancer. Tissue Antigens 61 (5): 335-43. doi:10.1034/j.1399-0039.2003.00070.x. PMID 12753652). However, because engineered soluble α1-α2 domains fused to heterologous polypeptides in certain embodiments of the instant invention reversibly bind specific target antigens on the surface of a target cell, the binding affinity of the engineered ULBP α1-α2 domains to NKG2D will directly affect the stability of the artificial synapse formed between NK cells and cells expressing target antigens, as already shown by engineered soluble MIC proteins (Examples 2-4). In order to diversify the repertoire of engineered non-natural α1-α2 domains as NKG2D ligands, ULBP proteins were used as a substrate or starting point for phage display-based engineering of their NKG2D binding affinity. Despite the structural homology observed between ULBPs and MICA (Radaev, S., Rostro, B., Brooks, A G., Colonna, M., Sun, P D. (2001) Conformational plasticity revealed by the cocrystal structure of NKG2D and its class I MHC-like Ligand ULBP3. Immunity 15, 1039-49.), the sequence homology is <50% for the ULBP α1-α2 domains relative to MICA (FIG. 18 ). Thus, we sought the identities of codon positions in ULBP α1-α2 domains that improve NKG2D binding affinity.

To engineer soluble, non-natural α1-α2 domains from ULBP proteins, ULBP2 and ULBP3 were chosen for phage display and selection of mutants with high affinity NKG2D binding. Sixty amino acid positions in the α1-α2 domain of ULBP2 (SEQ ID NO.:16), and thirty-six amino acid positions in the α1-α2 domain of ULBP3 (SEQ ID NO.: 17), were chosen for extensive mutagenesis (FIG. 18 ). In addition, conservative cysteine-to-serine mutations were made at C8S in ULBP2 (SEQ ID NO.:16) and C103S in ULBP3 (SEQ ID NO.: 17) in order to remove unpaired free cysteines that could interfere with phage panning. Synthetic DNA libraries coding for these α1-α2 domains, and containing NNK mutagenic codons at each of the selected amino acid positions, were synthesized, individually; cloned as fusions to the pIII minor coat protein of M13 phage; and phage particles displaying the mutagenized α1-α2 ULBP2 or ULBP3 variants were produced in SS320 E.coli cells according to standard methodologies (Andris-Widhopf, J., Steinberger, P., Fuller, R., Rader, C., and Barbas, C. F., 3rd. (2011). Generation of human Fab antibody libraries: PCR amplification and assembly of light- and heavy-chain coding sequences, Cold Spring Harbor protocols 2011). The α1-α2 phage display libraries were sorted for increased binding affinity to NKG2D using human NKG2D-Fc as the target protein, and cycled through iterative rounds of intentionally prolonged binding, prolonged washing, and eluting of the phage clones in order to select high affinity variants enriched for slow dissociation- or off-rates. For ULBP2, specific amino acid mutations were found at high frequencies at positions R80, V151, V152, and A153 in α1-α2, and were identified as preferred amino acid substitutions with enhanced NKG2D-binding affinity (FIG. 19A and Table 6).

Table 6. Selected affinity mutations at the indicated 4 amino acid positions of the α1-α2 domain of ULBP2. The amino acids of SEQ ID NOs.:16 at each of the 4 positions are shown in bold in the first row of the table. The identified affinity mutations are listed in decreasing frequency from top to bottom. All amino acids are represented by the single letter IUPAC abbreviations.

R80 V151 V152 A153 L D L E W E W K V Q G F K P I N S R A T E P T

For ULBP3, specific amino acid mutations were found at high frequencies in different locations relative to ULBP2 (FIG. 18 ). Positions R162 and K165 in the α1-α2 domain of ULBP3 contained specific mutations that were identified as preferred amino acid substitutions with enhanced NKG2D-binding affinity (FIG. 19 B and Table 7). These modified non-natural α1-α2 domains derived from ULBP2 and ULBP3 can be used for enhanced NKG2D binding in multiple therapeutic formats as single proteins or fusions to heterologous peptides or polypeptides.

Table 7. Selected affinity mutations at the indicated 2 amino acid positions of the α1-α2 domain of ULBP3. The amino acids of SEQ ID NOs.:17 at each of the 2 positions are shown in bold in the first row of the table. The identified affinity mutations are listed in decreasing frequency from top to bottom. All amino acids are represented by the single letter IUPAC abbreviations.

R162 K165 G S A P Y A T H N Q G

Example 6 (Modified α1-α2 Domains fused to antibody peptides). These are examples of attaching antibody polypeptides to NKG2DLs which were modified to significantly enhance their binding affinity to the human NKG2D receptor. The α1-α2 domain of MIC proteins is an NKG2DL for the NKG2D receptor. Antibodies are highly stable glycoproteins made up of two large heavy chains and two small light chains (FIG. 1 ). The large amount of diversity that can be generated within the CDR regions of the variable domains allows for specific antibodies to be generated to specific antigen targets (Hozumi N, Tonegawa S (1976). “Evidence for somatic rearrangement of immunoglobulin genes coding for variable and constant regions”. Proc. Natl. Acad. Sci. U.S.A. 73 (10): 3628-3632. doi:10.1073/pnas.73.10.3628. PMC 431171. PMID 824647.) Antibodies have become a significant therapeutic platform for drug development and can mediate both target binding and neutralization, as well as modulate the immune system through complement and Fc receptor binding (Vidarsson, G., Dekkers, G., Rispens, T. (2014) IgG subclasses and allotypes: from structure to effector functions. Frontiers in Immunology 5, 520.). Prior to the present invention, there did not exist an IgG antibody format that can directly activate immune cells using non-natural α1-α2 domains that bind more tightly than native NKG2DLs to the NKG2D receptor. Previous work has demonstrated that the mouse NKG2D ligand, Rael beta, can be fused to an anti-Her2 antibody for use as an anti-tumor agent in mice (Cho, H M., Rosenblatt, J D., Tolba, K., Shin, S J., Shin, D., Calfa, C., Zhang, Y., Shin, S U. (2010) Delivery of NKG2D ligand using and anti-Her2 antibody-NKG2D ligand fusion protein results in an enhanced innate and adaptive antitumor response. Cancer Research 70, 10121-30.). However, mouse NKG2D ligands do not bind human NKG2D, and there are no natural human NKG2D ligands with high affinity to human and mouse NKG2D. Fusions between the engineered α1-α2 NKG2D ligands of the instant invention and the heavy chain or light chain of IgG antibodies (FIGS. 20A and 20B) overcame these limitations and highlighted the versatility of fusions of modified α1-α2 domains to heterologous proteins or peptides.

To generate variant α1-α2 domain fusions to antibodies, the DNA sequences encoding α1-α2 domain for MIC WT, variants WED, 25, and 48, were synthesized and cloned as C-terminal fusions to either the heavy chain (HC_WT, HC_WED, HC_25, HC_48) or light chain (LC_WT, LC_WED, LC_25, LC_48) sequence from the FGFR3-specific antibody (Qing, J., Du, X., Chen, Y., Chan, P., Li, H., Wu, P., Marsters, S., Stawicki, S., Tien, J., Totpal, K., Ross, S., Stinson, S., Dornan, D., French, D., Wang, Q. R., Stephan, J. P., Wu, Y., Wiesmann, C., and Ashkenazi, A. (2009) Antibody-based targeting of FGFR3 in bladder carcinoma and t(4;14)-positive multiple myeloma in mice, The Journal of clinical investigation 119, 1216-1229.) (SEQ ID NOs.: 42-49, respectively). The resulting fusions were cloned into the mammalian expression vector pD2509 and expressed as paired full IgG antibodies with either heavy or light chain fusions of the modified α1-α2 domains (SEQ ID NOs.: 50-57, respectively). Transient expressions were carried out in HEK293 cells using the Expi293 expression system according to the manufacturer's protocol (Life Technologies), and purified using standard protein A affinity chromatography. The ability of the non-natural α1-α2-antibody fusions to redirect NK cell-mediated lysis of FGFR3-expressing target cells was demonstrated in vitro in a calcein-release assay. The human Natural Killer (NK) cell line, NKL, was co-cultured with calcein-loaded P815 target cells ectopically expressing FGFR3 and titrated with the engineered antibody fusion proteins. The results in FIGS. 20C and 20D showed that the killing activities of the FGFR3-specific non-natural α1-α2-antibody fusions correlated with their engineered NKG2D affinities. Specifically, antibodies that contained either heavy chain or light chain fusions of non-natural variants 25 and 48 (HC_25, HC_48 and LC_25, LC_48) killed FGFR3-expressing cells more effectively than antibody fusions containing either WT or WED α1-α2 domains.

This was further demonstrated to be a general and useful approach to fusing modified α1-α2 domains to antibodies, by fusing the variant 25 α1-α2 domain to the C-terminal of either the heavy chain or light chain of EGFR-specific antibody cetuximab (U.S. Pat. No. 6,217,866), Her2-specific antibody trastuzumab (Carter, P., Presta, L., Gorman, CM., Ridgway, JB., Henner, D., Wong, WL., Rowland, AM., Kotts, C., Carver, ME., Shepard, HM. (1992) Proc Natl Acad Sci 15, 4285-9), or an anti-PDL1 antibody (US Patent 20140341917) (SEQ ID NOs.:58-63, respectively). The resulting fusions were expressed as paired light and heavy chain full IgG antibodies with either heavy or light chain fusions of the variant 25 α1-α2 domain. Transient expressions were carried out in HEK293 cells using the Expi293 expression system according to the manufacturer's protocol (Life Technologies), and purified using standard protein A affinity chromatography. The ability of the variant 25 antibody fusions to redirect NK cell-mediated lysis of target-expressing cells was demonstrated in vitro in a calcein-release assay. The human Natural Killer (NK) cell line, NKL, was co-cultured with calcein-loaded A431 EGFR-expressing target cells, SKBR3 Her2-expressing target cells, or PDL1-expressing B16 cells and titrated with the respective target-specific engineered antibody fusion proteins. The results in FIGS. 21A, 21B, and 21C showed that the killing activities of the target-specific variant 25-antibody fusions were in all cases drastically improved over the non-fused parent antibody and very potent with sub-nanomolar EC50 values. These data show that modified α1-α2 variant-antibody fusions are a universal platform for allowing IgG antibodies to bind tightly to NKG2D and to direct antigen-specific cell lysis.

Example 7 (trastuzumab fusions to α1-α2 variant 25 bind NK cells in vivo and elicit potent antigen presentation). Fusion proteins containing α1-α2 domain variants that bind NKG2D with high affinity bound NK cells in vivo. Thus, antigen-specific antibodies containing modified α1-α2 fusions bind NKG2D tightly and thereby effectively armed the surface of NK cells in vivo with antibodies to seek out target cells expressing a particular antigen. This activity was similar to engineered CAR cells (Gill, S., and June, C H. (2015) Going viral: chimeric antigen receptor T-cell therapy for hematological malignancies. Immunol Rev 263, 68-89.), but did not require genetic modification of the NKG2D-expressing cell type.

To demonstrate that antibodies containing modified α1-α2 fusions bind NK cells in vivo, trastuzumab and the corresponding heavy and light chain fusions of variant 25 were analyzed in vivo for serum pharmacokinetic (PK) profiles and the pharmacodynamics (PD) of NK cell labeling. All three antibodies: parent trastuzumab; trastuzumab HC_25 fusion; and trastuzumab LC_25 fusion, were conjugated with Alexa Flour 647 according to the manufacturer's protocol (Life Technologies). Groups of three C57BL/6 mice were injected with a single dose of 100 μg of each antibody, and blood was drawn at indicated time points for plasma PK ELISAs and flow cytometry of peripheral NK cells. The PK profile of the parent trastuzumab antibody displayed typical alpha-phase distribution within 24-hrs and beta-phase elimination consistent with greater than a 1 week half-life of antibodies in mice (FIG. 22A). For both the heavy chain and light chain fusions with variant 25, the initial alpha-phase showed a much greater volume of distribution relative to the parent antibody, consistent with an NKG2D-sink, while the beta-phase elimination was also consistent with typical antibody clearance in mice (FIG. 22A). Using flow cytometry of peripheral NK cells from the mouse blood, the level of NK cell staining with Alexa Fluor 647 showed a clear time-dependent increase in the percent of NK cells labeled with the antibody fusion, but not the parent antibody (FIG. 22 B). The increase in labeling by the fusions peaked within 24 hrs, consistent with the sink observed in the PK profiles for the fusions, and was stable at least for three days post injection. The combined PK and PD data demonstrate that the trastuzumab antibodies containing variant 25 α1-α2 fusions formed stable complexes with NKG2D on NK cells in vivo.

To assess the appearance of anti-drug antibodies (ADAs) to the human IgG trastuzumab antibody, the plasma samples from the PK/PD study were assessed for ADAs using an ELISA. In FIGS. 23A-C, ELISAs for mouse IgG binding to wells coated with the 3 respective dosed antibodies revealed that only the antibodies fused with variant 25 elicited ADAs within seven days after a single dose of antibody. The parent trastuzumab antibody gave no ADA signal. In order to determine whether the antibody fusions elicited an immune (ADA) response to both the α1-α2 domain and the antibody (trastuzumab) component when the trastuzumab antibody itself did not elicit an immune response, the ADA-positive plasma from the antibody fusions were tested against the parent antibody and the variant 25 α1-α2 domain individually; both moieties reacted with ADAs from plasma (FIGS. 24A and 24B). These data demonstrate that the fusion of high affinity variant 25 to the parent antibody mediated NKG2D-dependent uptake and antigen presentation to elicit potent and rapid immune responses to the parent antibody, which alone was not so immunogenic in mice. Thus, a high affinity variant α1-α2 domain attached to an antigen or immunogen provided potent presentation of the antigen and epitope spreading, effectively serving as a potent adjuvant for immunization.

The demonstrated combined effects of arming circulating NK cells for directed target cell lysis and enhancing antigen presentation are important activities for antibody fusions to modified α1-α2 domains that can provide therapeutic benefit.

Example 8 (Antibody heavy chain fusion to α1-α2 variant 25 exhibited anti-tumor activity in vivo). To examine the potential for antigen-specific antibodies fused to modified α1-α2 to have anti-target cell activity, an anti-PDL1 antibody heavy chain fusion to variant 25 α1-α2 was evaluated in a syngeneic MC38 tumor model. MC38 tumors were implanted sub-cutaneously in C57BL/6 mice and tumors grew to an average of 100 mm³ before the initiation of treatment. Upon initiation of treatment, four cohorts of 10 mice per group were treated with vehicle, anti-CTLA4 (100 ug i.p.), parent anti-PDL1 (300 ug i.v.), or anti-PDL1 HC_(˜)25 fusion (300 ug i.v.) on days 1, 4, and 7. In FIG. 25 , the tumor growth curves showed that anti-PDL1 HC_(˜)25 mediated the most effective anti-tumor activity within the first two weeks of treatment. Tumor growth inhibition was significantly better than the established anti-CTLA4 treatment and the parent anti-PDL1 antibody over the first 12 days after initiation of treatment. By day 16, the anti-PDL1 HC_25 treatment began to lose efficacy consistent with the occurrence of an ADA response as observed for trastuzumab fusions (Example 7). The significant anti-tumor activity observed for the antibody heavy chain fusion to variant 25 relative to both the parent antibody and standard anti-CTLA4 treatments demonstrated the impressive therapeutic effect of antibody fusions to modified α1-α2 domains that served as high affinity NKG2D ligands. 

What is claimed is:
 1. An insertable Fv (iFv) polypeptide exhibiting specific antigen-binding properties comprising a variable fragment (Fv) of an antibody, wherein the specific antigen-binding domain of the Fv comprises two antibody variable domains, one variable domain from the light chain (VL) and one variable domain from the heavy chain (VH), each with 1-3 complementarity determining regions (CDRs), and each with a C-terminus and an N-terminus; and wherein the VL and VH domains are joined by two linker regions connecting both canonical termini of the VL domain to both canonical termini of the VH domain.
 2. The iFv of claim 1, wherein a non-natural pair of N- and C-termini have been created within either the VL or the VH domain to provide an attachment site for conjugation to or insertion into a recipient molecule.
 3. The iFv of claim 2, wherein the non-natural N- and C-termini are spatially positioned proximal to each other.
 4. The iFv of claim 2, further comprising a recipient polypeptide or protein containing one or more solvent exposed loops, wherein the iFv is inserted into one or more of the loops.
 5. The iFv of claim 2, further comprising a recipient polypeptide or protein containing one or more solvent exposed loops, wherein the iFv is inserted into one or more of the loops, such that the non-natural N- and C-termini within the VL or VH domains are spatially positioned proximal to each other.
 6. The iFv of claim 1, wherein either the C-terminus of the VL domain is connected to the N-terminus of the VH domain and the N-terminus of the VL domain is connected to the C-terminus of the VH domain; or wherein the N-terminus of the VL domain is connected to the C-terminus of the VH domain and the C-terminus of the VL domain is connected to the N-terminus of the VH domain.
 7. The iFv of claim 5, wherein the distance between the non-natural N- and C-termini is between about 0.2 and about 2.0 nm.
 8. The iFv of claim 7, wherein the distance between the termini is between about 0.5 nm and about 1.5 nm.
 9. The iFv of claim 1, wherein the two linker regions comprise identical or non-identical peptides of about 10 to 25 amino acids.
 10. The iFv of claim 5, wherein the recipient polypeptide or protein is an antibody, Ig fold, Ig domain, globulin, albumen, fibronectin, fibronectin domain, integrin, fluorescent protein, enzyme, outer membrane protein, receptor protein, T-cell receptor, chimeric antigen receptor, viral antigen, virus capsid, viral ligand for cell receptor, high molecular weight bacteriocin, histone, hormone, knottin, cyclic peptide, ULB protein, lectin, or a ligand for a lectin.
 11. The iFv of claim 1, further comprising a non-protein molecule to which the iFv is inserted into, or attached.
 12. The iFv of claim 11, wherein the non-protein molecule is a polysaccharide, dendrimer, polyglycol, peptidoglycan, chemotherapeutic agent, toxin, fluorophore, chromophore, pharmacophore, antibiotic or polyketide.
 13. The iFv of claim 2, further comprising a non-protein molecule or an atom conjugated to one or both of the non-natural N-terminus and the non-natural C-terminus.
 14. The iFv of claim 5, wherein all or a portion of the one or more solvent-exposed loops of the recipient molecule is deleted and replaced with the iFv. 